Current models of carcinogenesis describe cancer as a progression of genetic mutations in a tumour cell mass and these models have contributed to the discoveries of many tumour suppressor genes and potential oncogenes (Hanahan D. et al. Cell 100:57 (2000)). The progression of genetic mutations can arise from a genetic instability in the cell leading to a loss in replication fidelity, genetic translocations or loss of genetic material. Solid tumours, however, are more than clonal expansions of tumour cells; tumours are heterogeneous and have a complex structure, with Bissell et al. describing a tumour as a unique “organ” formed by “tissues” (Bissell, M. J. et al. Nat Rev Cancer 1:46 (2001)). The cells composing these tissues interact with each other and with other types of cells, and exchange information through cell-cell interactions or through interactions with cytokines and the extracellular matrix (ECM) (Bissell, M. J. et al. Nat Rev Cancer 1:46 (2001)). Playing an important role in these interactions, and possibly playing a role in proliferative disease progression as taught in the art and as discovered by the inventors and herein disclosed, is hyaluronic acid (hereinafter “HA”).
HA, a non-sulfated negatively charged glycosaminoglycan, is composed of repeating disaccharide units of D-glucorinic acid and N-acetylglucosamine. HA is completely biodegradable by a natural catalytic pathway and is widely distributed in all connective tissue of eukaryotes and in the capsules of group A and C streptococci (Laurent, T. C. et al. FASEB J. 6:2397 (1992)). HA is involved in many biological processes such as embryogenesis, cell adhesion and motility, cell growth and differentiation, and angiogenesis (Baneijee, S. D. et al. J Cell Biol 119:643 (1992); Bourguignon, L. Y. et al. J Biol Chem 272:27913 (1997); Lees, V. C. et al. Lab Invest 73:259 (1995); West, D. C. et al. Science 228:1324 (1985)).
HA, which is widely distributed in all connective tissue of eukaryotes, is a water-like molecule; because of this characteristic HA has been regarded as an ideal lubricant of the joints and has been successfully used in the treatment of patients with arthritis (Radin, E. L. et al. Nature 228:377 (1970)), where HA forms a layer between the cartilage surfaces in joints and protects them from frictional damage (Hlavacek, M., J Biomech 26:1151 (1993)). In arthritis, the mechanism forming protective HA layers is disrupted since the concentration of HA itself and molecular weight of the HA molecules are low as compared to normal tissues (Hlavacek, M., J Biomech 26:1151 (1993)). Depletion of HA results in degradation of the ECM and promotes osteoarthritis, a degenerative disease of articular cartilage.
Dramatically increased HA-rich matrix formation has been observed around proliferating and migrating cells during morphogenesis, regeneration and healing. High amounts of HA molecules are synthesized:                1) prior to the mesenchymal cell differentiation and throughout embryonic development, the condensation and differentiation of the mesenchymal cells are accompanied by the spatial distribution of HA in the different regions of the limb bud (Kosher, R. A. et al. Cell Differ 17:159 (1985); Kosher, R. A. et al. Nature 291:231 (1981); Kosher, R. A. et al. J Embryol Exp Morphol 56:91 (1980));        2) during brain development around proliferating and migrating neuronal cells, (Verna, J. M. et al. Int J Dev Neurosci. 7:389 (1989)); and        3) during formation of heart valves when cushion cells migrate from the endocardium to the myocardium (Camenisch, T. D. et al. J Clin Invest 106:349 (2000)).        
HA matrices are removed from the cells after final differentiation at the end of morphogenetic events (Gakunga, P. et al. Development 124:3987 (1997)). Throughout morphogenesis HA creates hydrated pathways, thus facilitating free movement of the cells in this microenvironment (Gakunga, P. et al. Development 124:3987 (1997)). HA molecules are conducive to cell proliferation and migration, preventing differentiation of cells until sufficient number and appropriate positioning of cells is established, which is essential for the formation of tissues and/or organs (Gakunga, P. et al. Development 124:3987 (1997)). In addition, the formation of hydrated pathways by HA molecules is closely associated with the surface of different types of cells, and these associations promote cell adhesion and aggregation (Sionov, R. V. et al. Adv Cancer Res 71:241 (1997); Lee, V. et al. J Cell Biochem 79:322 (2000)).
The motility of malignant cells is mediated through interactions with HA, which is an important extracellular matrix molecule (Docherty, R. et al. J Cell Sci 92:263 (1989); Ropponen, K. et al. Cancer Res 58:342 (1998); Ruoslahti, E. J Biol Chem 264:13369 (1989); Sherman, L. et al. Curr Opin Cell Biol 6:726 (1994); Zhang, W. et al. Biochem J 349:91 (2000)). High or very low levels of HA in the serum of patients with multiple myeloma (MM) correlate with dramatically reduced median survival of these patients (Dahl, I. M. et al. Blood 93:4144 (1999b)). Moreover, HA mediates survival of MM cell lines against dexamethasone-induced apoptosis through IL-6-dependent and -independent autocrine pathways (Vincent, T. et al. Br J Haematol 121:259 (2003)). HA also increases intracellular Ca2+ levels by binding to CD44, suggesting that HA may activate intracellular signaling through activation of protein kinase C (Fraser, S. P. FEBS Lett 404:56 (1997); Liu, D. et al. Cell Immunol 174:73 (1996); Milstone, L. M. et al. J Cell Sci 107:3183 (1994)). Also secretion of HA is stimulated by growth factors which activate classical and novel isoform (PKCa) of PKC (Anggiansah, C. L. et al. J Physiol 550:631 (2003)). In addition to its role as an ECM and signaling molecule, HA plays a significant role in the process of mitosis and in the maintenance of cell shape or volume (DeAngelis, P. L., Cell Mol Life Sci 56:670 (1999); Evanko, S. P. et al. Arterioscler Thromb Vasc Biol 19:1004 (1999)).
Biochemical and cell biological studies suggest that HA is synthesized at the inner face of the plasma membrane and is immediately extruded into the extracellular matrix (ECM) where HA molecules are assembled and form a pericellular coat around the cell plasma membrane (Weigel, P. H. et al. Biol Chem 272:13997 (1997)). The manner of the synthesis of HA molecules is unique and differs from the synthesis of other glycosaminoglycans that occurs at the golgi, with finished products exported to the cell surface by proteins. In addition to extracellular HA, an intracellular HA derived from an intracellular pool in the cell has also been detected (Evanko, S. P. et al. Histochem Cytochem 47:1331 (1999)). The production of HA is stimulated by phobol esters, the transforming growth factor beta (TGF-β), and the platelet-derived growth factor (PDGF) family, by activation of protein kinase C and cAMP (Honda, A et al. Biochem J 292:497 (1993); Pienimaki, J. P. et al. J Biol Chem 276:20428 (2001); Suzuki, M. et al. Biochem J 307:817 (1995)).
Hyaluronan synthase (HAS), is an integral membrane protein which mediates 7 distinct functions in order to assemble and translocate HA molecules through the cell plasma membrane. The HAS protein is associated with malignant cell transformation (Banerjee, S. D. et al. J Cell Biol 119:643 (1992); Suzuki, M. et al. Biochem J 307:817 (1995)). The activation of HAS and consequently HA production strongly correlates with the transforming activity of v-src (Sohara, Y. et al. Mol Biol Cell 12:1859 (2001)). Furthermore, tumour-specific activation of cell migration is mediated by two parallel pathways the Ras-MAPK and the PI3K-Akt pathway (Sohara, Y. et al. Mol Biol Cell 12:1859 (2001)).
Heldermon et al. proposed an updated topological structure of Streptococcus pyogenes hasA (sphasA) (Heldermon, C. D. et al. Glycobiology 11:1017 (2001)). Their proposed structure includes four transmembrane domains (TDM), two extracellular loops, two membrane-associated regions, an intracellular central loop, and intracellular amino and carboxyl terminals. Due to their extensive primary sequence identity, it has been proposed that eukaryotic and bacterial HAS proteins have the same topological organization and they belong to the same class, class 1, of HAS protein family (Table 1) (DeAngelis, P. L., Cell Mol Life Sci 56:670 (1999)). The eukaryotic HAS is 40% larger than bacterial HAS and its topology includes an additional trasmembrane domain and an extracellular loop.
TABLE 1Classes of HAS proteins (DeAngelis,P. L., Cell Mol Life Sci 56:670 (1999))Class IClass IIMembersSpHas, seHas, cvHas, xlHas,PmHasvertebrate HAS1, 2, 3Polypeptide Size417-588 residues972 residuesTopologymultiple pass, integralsoluble with a dockingmembrane proteinsegment for membranepartner
Examination of the hydrophobic domains of HAS isoenzyme variants have shown the existence of cysteine residues in the amino acid sequences of the central domain (Heldermon, C. et al. J Biol Chem 276:2037 (2001)). Modification of these cysteine residues changes the enzymatic activities of the HAS proteins (Heldermon, C. et al. J Biol Chem 276:2037 (2001); Pummill, P. E. et al. J Biol Chem 278:19808 (2003)). Recently, it has been shown that single amino acid mutation introduced on the HAS protein alters the size of the HA molecules, specifically the size of HA chain can be either reduced or increased (Pummill, P. E. et al. J Biol Chem 278:19808 (2003)). The serine, tyrosine, and cysteine(s) which are responsible for this process are conserved and located either on the central loop (tyrosine, and cysteine(s)) while serine is part of the TMD of the protein. A significant number of protein kinase C phosphorylation sites are predicted within the intracellular loop of the HAS protein suggesting that HAS activation is perhaps regulated by direct phosphorylation, and suggesting potential sites for therapeutic attack.
Three isoenzymes of HAS: HAS1, HAS2, and HAS3; have been detected in humans thus far. The related but separate genes of the HASs, which share at least one or two exon-intron boundaries and 55-71% amino acid sequence identity, are located on different chromosomes (hCh19-HAS1, hCh8-HAS2, hCh16-HAS3) and encode three different proteins with distinct enzymatic properties (Itano, N. et al. J Biol Chem 274:25085 (1999); Spicer, A. P. et al. Genomics 41:493 (1997)). Recently two variants of HAS3, HAS3vl and HAS3v2, have been reported (NCBI database). The similarities of gene structure suggest that these genes might have arisen by a gene duplication event (Spicer, A. P. et al. Genomics 41:493 (1997)). Also localization of HASs in different chromosomes suggests differential expression of these genes and not completely similar functions. Each isoform of the HAS protein synthesizes different sizes of HA molecules with different functions. HAS3 synthesizes shorter forms of HA molecules compared to HAS1 and HAS2, both of which produce longer molecules of HA. The HAS1, which synthesizes high-molecular weight HA, may maintain a low, basal level of HA. HAS2 is involved in embryonic and cardiac cushion morphogenesis and subsequent development through cell migration and invasion (Camenisch, T. D. et al. J Clin Invest 106:349 (2000)). This form of HA induces cell proliferation and activates cell signaling cascades which stimulate angiogenesis.
Overexpression of HAS proteins and subsequent overproduction of HA molecules promotes growth and/or metastatic development in fibrosarcoma, prostate and mammary carcinoma. However, the removal of the HA matrix from a migratory cell membrane inhibits cell movement, as has been demonstrated by Baneijee et al. and Evanko et al. (Banedji, S. et al. J Cell Biol 144:789 (1999); Evanko, S.P. et al. Arterioscler Thromb Vasc Biol 19:1004 (1999)). HAS proteins, particularly the HAS2 isoenzyme, which is involved in embryonic and cardiac cushion morphogenesis and subsequent development through cell migration and invasion, appear to facilitate abnormal cell proliferation and the activation of cell signaling cascades that stimulate angiogenesis and may promote tumour progression (Lees, V.C. et al. Lab Invest 73:259 (1995); West, D. C. et al. Science 228:1324 (1985)).
HA overproduction by cushion cells not only provides a substrate for cardiac cell migration but also influences the transformation of these cells into a motile phenotype, suggesting a significant role in oncogenesis (Lees, V. C. et al. Lab Invest 73:259 (1995); Li, H. et al. Int J Oncol 17: 927 (2000)). HA is produced in large quantities by cells undergoing mitosis as well. It facilitates cell rounding and is involved in the post-mitotic separation of daughter cells (Evanko, S. P. et al. J Histochem Cytochem 47:1331 (1999); Tammi, R. et al. Exp Cell Res 195:524 (1991)). The activation of HAS enzymes appears to be essential for these events. The expression of the antisense of HAS2 and/or HAS3 in the aggressive prostate adenocarcinoma PC3M-LN4 cell line inhibited tumour growth (Liu N. et al. Cancer Res 61:5207 (2001)). This finding suggests that overproduction of HA is required for tumour progression and it appears that elevated production of HA by prostate stroma and cancer cells is a negative prognostic factor (Liu, N. et al. Cancer Res 61: 5207 (2001)). Overexpression of HAS2 and HAS3 promotes anchorage-independent growth and tumourigenicity in immunocompromised mice (Kosaki, R. et al. Cancer Res 59:1141 (1999); Liu, N. et al. Cancer Res 61:5207 (2001); Li, Y. et al. Br J Cancer 85:600 (2001)). Compared to HAS1 and HAS3, the HAS2 gene is easily regulated in response to mechanical injury in human peritoneal mesonthelial cells in vitro and in dermal fibroblasts and osteoblasts in response to glucocorticoids (Yung, S. et al. Kidney Int 58:1953 (2000); Jacobson, A. et al. Biochem J 348:29 (2000); Zhang, W. et al. Biochem J 349:91 (2000)). Transfected HAS2 has been shown to induce transformed growth (Zoltan-Jones, A. et al. J Biol Chem 2003 Sep 3 [Epub ahead of print])
Overexpression of HAS1 or HAS2, both of which appear to synthesize high molecular weight HA, may activate hyaluronidase, an enzyme which degrades HA molecules and which is observed to be upregulated or downregulated during the progression of human cancer (Lees, V. C. et al. Lab Invest 73:259 (1995)). Shorter forms of HA resulting from HA degradation have been implicated in angiogenesis (Lees, V. C. et al. Lab Invest 73:259 (1995); West, D. C. et al. Science 228:1324 (1985); West, D. C. et al. Ciba Found Symp 143:187 (1989)). Therefore, changes to HAS1 and/or HAS3 gene regulation most likely results from significant changes in the cell or tissue in response to external or internal stimuli.
In mammary carcinoma cells, transfection with HAS1 transcripts resulted in the formation of increased metastasis as compared to that in controls (Itano, N. et al. Cancer Res 59:2499 (1999)). However, little is known about the role of HAS1 in various types of cancers, and regulation in response to external stimuli is not clear because of the nature of the gene. The lifetime of HAS1 transcripts may be very short and/or HAS1 may be expressed at low levels, compromising its detection by standard conventional gel electrophoresis. However, since HAS1 appears to be stringently regulated, its basal overexpression by tumour cells appears to be the result of dramatic changes in these cells.
In addition to extracellular HA molecules which are extruded into the extracellular compartment, an intracellular HA has been detected in the cytoplasm and the nucleus of various tissues such as the brain, liver, arteries, cumulus cells and oocytes (Dahl, I. M. et al. Blood 93:4144 (1999a); Furukawa, K. et al. Biochim Biophys Acta 585:575 (1979); Itano, N. et al. Cancer Res 59:2499 (1999); Londono, I. et al. Histochem Cytochem 36:1005 (1988); Margolis, R. K. et al. Biochim Biophys Acta 451:465 (1976); Ripellino, J. A. et al. J Cell Biol 106:845 (1988); Ripellino, J. A. et al. J Cell Biol 108:1899 (1989); Simpson, M. A. et al. Am J Pathol 161:849 (2002)). This intracellular HA can regulate gene transcription and/or the cell cycle by binding to the cell cycle control protein CDC37 and through the activation of the erk kinase pathway via the intracellular form of the HA binding receptor, Receptor for HA Mediated Motility (RHAMM) (Grammatikakis, N. et al. J Biol Chem 270:16198 (1995); Zhang, S. et al. J Biol Chem 273:11342 (1998)). In turn, activation of the erk kinase could induce transcription of many types of genes.
Intracellular localization of HA has been detected in nucleoli and in the nuclear periphery of areas of condensed chromatin (Evanko, S. P. et al. J Histochem Cytochem 47:1331 (1999)). Evanko et al. showed localization of intracellular HA during mitosis at the metaphase plate (Evanko, S. P. et al. J Histochem Cytochem 47:1331 (1999)). Furthermore, intracellular HA was detected around chromosomes during their rearrangement and separation in anaphase. In concert with previous findings, this work suggests a significant role of HA and HASs in mitosis and especially in chromatin condensation which occurs through interaction with histone and lamin (Cremer, T. et al. Nat Rev Genet 2:292 (2001)). Evanko et al. suggest a possible role for nuclear HA in ribosomal production and trafficking, or MRNA processing (Evanko, S. P. et al. J Histochem Cytochem 47:1331 (1999)). The source of intracellular HA, up to now, has remained unclear.
Multiple Myeloma (MM) is an incurable bone marrow (BM) cancer characterized by osteolytic bone lesions, BM plasmacytosis and monoclonal gammopathy. Molecular studies conducted in our laboratory revealed circulating, late stage, drug-resistant B-cells with clonotypic VDJ rearrangements in the peripheral blood (PB) of patients with MM. We believe that these cells migrate to the BM and give rise malignant plasma cells (PC) subsequently facilitating disease progressions. These drug-resistant clonotypic cells express the RHAMM oncogene. Furthermore, over-expression and/or inhibition of intracellular RHAMM dysregulates mitosis, likely leading to chromosomal instabilities and malignant spread in MM by mediating HA-dependent motility. Given the high remission rate and poor understanding of the disease, clinicians face a difficult challenge in directing treatment for the disease. A diagnostic test allowing the clinician to assess the severity of the disease through assessment of the likelihood of patient survival would be of great assistance to the medical community.
Current methods for monitoring myeloma involve bone marrow aspirates or core biopsies of the bone marrow, painful procedures that require an MD to perform them. The cells in the aspirate or biopsy are then viewed by an experienced pathologist using microscopic analysis, a time consuming process. This mode of testing depends on the biopsy needle penetrating an area of the bone marrow that is infiltrated by malignant cells. Since the distribution of such cells is not even throughout the bone marrow, this can result in “geographic misses” when the needle draws cells from an area that does not have malignant infiltration. It is also insensitive in that it cannot detect cells that have morphology different from that of classical plasma cells even though such cells may have the molecular signature that unequivocally identifies the myeloma clone. An alternate method involves analysis of monoclonal protein, the protein secreted by the bone marrow plasma cells, as measured by a blood test. This test is relatively painless but is insensitive, able to detect only relatively high levels of the protein excluding its use for monitoring minimal disease and early stages of relapse, and does not detect those patients whose cancer does not secrete the protein or as disease progression occurs has lost the ability to secrete it.